PROJECT SUMMARY. We propose that there is a biological switch in estrogen receptor action that occurs coincident with age and length of time after ovarian hormone depletion (i.e. menopause). Accumulating evidence suggests that ERb shifts from a predominantly ligand-activated transcription factor (major role during reproductive years) to a ligand-independent transcription factor post-menopause. Elucidating the molecular basis for this shift in signaling paradigms is critical for understanding clinical observations, which demonstrate a defined narrow window of time for therapeutic efficacy of hormone therapy in postmenopausal women. This application is a competitive renewal of R01 AG033605. Our previous aims for this project centered on factors that we predicted would differentially dictate a dominant ligand-dependent signaling pattern for ERb, as opposed to a ligand-independent signaling pattern. Through that work, we identified 3 key molecular factors that facilitated ligand-independent function of ERb in the aged brain: receptor phosphorylation, alternative RNA splicing, and coregulatory protein interactions. These mechanisms represented both direct changes to the receptor itself and indirect changes in protein:protein interactions. The experiments proposed in this renewal will be focused on the direct changes to the ERb protein itself, namely phosphorylation and alternative splicing. Aim 1 will focus on posttranslational phosphorylation of the receptor. Phosphorylation of ERb is a strong facilitator of ligand-independent activity, yet the extent of phosphorylated ERb present in the aged female brain, and the in vivo functional consequences of such a modification, represents a major gap in our current knowledge. We propose a comprehensive proteomics approach to a) quantify phosphor-ERb in the aged female brain, b) determine how site-specific phosphorylation alters ERb protein:protein interactions, and c) identify specific phospho-ERb target genes. Aim 2 will focus on alternative RNA splicing of the nascent ERb transcript. Our data and others have demonstrated the functional significance of ERb splice variants and we have shown that these alternative variants increase during aging. However, we lack a fundamental understanding of the molecular mechanisms regulating ERb splicing in the brain, or in any tissue type. We have now identified several putative splicing factors that regulate alternative splicing of ERb in the aged brain. This aim will test the direct effects of these splicing factors on ERb alternative splicing and also determine if E2 regulates global splicing events by modulating the expression of these splicing factors. Our preliminary data also showed that transcriptional kinetics play an important role in ERb splicing, with slower transcription favoring exon inclusion resulting in increased ERb2. This aim will further explore this relationship to test how transcriptional kinetics in the brain change in our aging female rat menopause model. Impact: Understanding the basic molecular signaling pathways of E2 in the aging brain will help drive therapeutic advances and inform treatment strategies for postmenopausal women.